Volume 154, Issue 5, Pages 1023-1035 (August 2013) Serotonin and the Neuropeptide PDF Initiate and Extend Opposing Behavioral States in C. elegans  Steven W. Flavell, Navin Pokala, Evan Z. Macosko, Dirk R. Albrecht, Johannes Larsch, Cornelia I. Bargmann  Cell  Volume 154, Issue 5, Pages 1023-1035 (August 2013) DOI: 10.1016/j.cell.2013.08.001 Copyright © 2013 Elsevier Inc. Terms and Conditions

Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Serotonin Affects Exploration Behavior (A) Locomotion of an adult wild-type animal on an E. coli lawn. Locomotion speed and angular speed (turning rate) differ in two distinct behavioral states, roaming and dwelling. (B) Simplified assay for measuring exploration behavior based on movement across a 35 mm bacterial lawn. The grid has 86 squares. The exact number of squares entered can vary from day to day, so all genetic manipulations are compared to controls tested in parallel. (C) Exploration behavior of 57 mutant strains normalized to wild-type controls. Asterisks indicate statistical significance at FDR < 0.05. For detailed results, see Table S1. (D) Rescue of mod-1 mutant phenotype by a mod-1::mod-1::GFP transgene. (E) Rescue of tph-1 mutant phenotype by a genomic tph-1 transgene. For (D and E), asterisks indicate p < 0.05 by ANOVA and Bonferroni-Dunn post hoc test. Data are shown as means ± SEM. See also Figure S1 and Table S1. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Behavioral State Defects in Serotonergic Signaling Mutants (A) Complementary cumulative distribution function (ccdf) for dwelling-state durations in wild-type animals (dots) fit to a single exponential (pink line). (B) Ccdf for roaming state durations in wild-type animals (dots) fit to a double exponential (pink line). The two exponentials are individually displayed as dashed black lines, and w1/w2 are the weights of each exponential, i.e., 85% of roaming states have a mean lifetime of 75 s. (C and D) Behaviors of wild-type and mutant animals. (C1 and D1) Fraction of time spent in roaming and dwelling states. (C2 and D2) Dwelling state durations and (C3 and D3) roaming state durations, expressed as means of the individual animal mean ± SEM, not as event distributions as in (A and B). Asterisks indicate p < 0.01, t test. See also Figure S2. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 A Distributed Serotonergic Circuit Controls Exploration Behavior (A and B) Cell-specific deletion of tph-1 using a Cre/Lox strategy. (A) Schematic depicting genotypes. (B) Exploration behavior. Asterisks indicate p < 0.01 (versus wild-type) by ANOVA with Dunnett test. (C–E) Intersectional cell-specific rescue of mod-1 using an inverted Cre-Lox strategy. (C) Schematic depicting transgenes. (D) Rescue of mod-1 by expression in AIY, RID, and ASI. (E) Rescue of mod-1 by expression in RIF. For (D and E), asterisks indicate p < 0.05 by ANOVA with Bonferroni-Dunn post hoc test. (F) Laser ablations of individual mod-1-expressing neurons. RID, PVN, and RIF were ablated in mod-1 mutants; AIY and ASI were ablated in wild-type animals. Asterisks indicate p < 0.05, t test. (G) Serotonin promotes dwelling by inhibiting MOD-1-expressing neurons that promote roaming. All data are shown as means ± SEM. See also Figure S3. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Changes in NSM and AIY Calcium Levels Correlate with Behavioral Transitions (A) Representative image from a video recording of a freely moving NSM::GCaMP5 transgenic animal on a bacterial lawn. Asterisk indicates the position of the NSM neuron; gut autofluorescence is also visible. (B) NSM calcium imaging in a freely moving animal as in (A). Arrows mark calcium peaks (see Extended Experimental Procedures for identification criteria). Roaming states are abbreviated in the calcium-imaging device due to the small viewing field and bacterial lawn; nevertheless, we observe clusters of forward runs that are related to roaming states. (C–F) Averaged NSM calcium levels, speeds, and forward runs in wild-type and mod-1 animals. (C and E) Event-triggered average aligning NSM calcium peaks with locomotion speed and forward runs in wild-type animals (C, n = 112; p < 0.001 for calcium levels and speed, before versus during peak, paired t test) and mod-1 mutants (E, n = 61; p < 0.001 for speed during calcium peak in wild-type versus mod-1, t test on values normalized to pre-event baseline; p < 0.001 for fraction of animals in forward run, wild-type versus mod-1, chi-square test). (D and F) Event-triggered average aligning forward runs with NSM calcium signal in wild-type animals (D, n = 51; p < 0.01 for NSM calcium during pre-event baseline versus forward run or −60 to −20 s, paired t test) and mod-1 mutants (F, n = 32; p < 0.01 for NSM calcium during pre-event baseline versus forward run, paired t test, no significant difference at −60 to −20 s). (G) Event-triggered average aligning forward runs with AIY calcium signal in wild-type animals (n = 27; p < 0.01 for AIY calcium during pre-event baseline versus forward run, paired t test). For (C–G), horizontal dashed lines indicate pre-event baseline calcium signals and speed. All data are shown as means ± SEM. See also Figure S4. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Optogenetic Manipulations of a Serotonergic Neural Circuit (A) ChR2-mediated activation of serotonergic neurons in animals that were roaming (red) prior to LED illumination. (B) ARCH-mediated silencing of serotonergic neurons in animals that were dwelling (blue) prior to LED illumination. (C) ARCH-mediated silencing of mod-1-expressing neurons in animals that were roaming prior to LED illumination. (D) ChR2-mediated activation of mod-1-expressing neurons in animals that were dwelling prior to LED illumination. For (A–D), asterisks indicate p < 0.001 compared to control strain, chi-square test, and controls (gray lines) were identically treated wild-type animals. Blue light delivery was followed by 60 s of green light delivery to inactivate ChR2(C128S). (E) Long-lasting effects of mod-1::ARCH activation. Left: experimental design. Right: durations of control dwelling states and dwelling states that overlapped with ARCH activation. Data are shown as medians ± 95% confidence intervals. Asterisk indicates p < 0.05, Wilcoxon rank-sum test. See also Figure S5. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 PDF Signaling Controls Exploration Behavior (A) Exploration behavior of PDF signaling mutants. ∗∗p < 0.01 and ∗∗∗p < 0.001 by ANOVA and Bonferroni-Dunn post hoc test. (B and C) Fraction of time spent in roaming and dwelling states and dwelling and roaming state durations in (B) pdfr-1 and (C) pdf-1; pdf-2 mutants, shown as in Figures 2C and 2D. Asterisks indicate p < 0.01, t test. (D and E) Cell-specific deletion of pdf-1 using a Cre/Lox strategy. (D) Schematic depicting transgenes. (E) Exploration behavior. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by ANOVA with Dunnett test. (F–H) Intersectional cell-specific rescue of pdfr-1 using an inverted Cre-Lox strategy. (F) Schematic depicting transgenes. (G) Rescue of pdfr-1 by expression in all pdfr-1-expressing neurons. (H) Partial rescue of pdfr-1 by expression in AIY, RIM, and RIA. For (G and H), asterisks indicate p < 0.01 by ANOVA and Bonferroni-Dunn post hoc test. All data are shown as means ± SEM. (I) Neural circuit for exploration behavior. Synapses and gap junctions in the C. elegans wiring diagram are in black; neuromodulatory connections defined here are in red (serotonin) and green (PDF). NSM neurons are implicated in feeding, and HSN neurons are implicated in egg laying. AIY and RIM neurons regulate reversal frequencies. ASI neurons are sensory neurons (triangles) that sense food, pheromones, and peptide cues to regulate dauer larva development. RIA interneurons regulate head curving during locomotion. AVB interneurons are forward command neurons in the motor circuit. PVP, SIAV (not diagrammed here), and RIF interneurons do not have other known functions. See also Figure S6. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 pdfr-1 Acts through cAMP Signaling and Relationship of Serotonin and PDF Signaling Pathways (A) Roaming and dwelling states in pdfr-1::acy-1(P260Sgf) animals, shown as in Figure 2C. (A1) Fraction of time spent in roaming and dwelling states. (A2) Dwelling state durations. (A3) Roaming state durations. Asterisks indicate p < 0.01, t test. (B) Exploration behavior of pdfr-1::acy-1(P260Sgf) animals. Asterisks indicate p < 0.01, ANOVA and Bonferroni-Dunn post hoc test (versus wild-type). (C) Optogenetic strategy for mimicking acute PDFR-1 activation. (D) BlaC activation in pdfr-1-expressing neurons in animals that were dwelling prior to LED illumination. Controls (gray line) were identically treated wild-type animals. Asterisks indicate p < 0.05, chi-square test. (E) Roaming and dwelling in single and double mutants. (E1) Fraction of time spent in roaming and dwelling states. (E2) Dwelling state durations. (E3) Roaming state durations. Asterisks indicate p < 0.05, ANOVA and Bonferroni-Dunn post hoc test (for E3, each genotype compared to wild-type). (F) pdfr-1::BlaC activation induces roaming behavior in mod-1; pdfr-1 double mutants that were dwelling at the time of LED illumination. (G) mod-1::ARCH activation induces dwelling behavior in mod-1; pdfr-1 double mutants that were roaming at the time of LED illumination. Controls (gray lines) were matched mutant animals with no LED illumination. Asterisks indicate p < 0.01, chi-square test. All data are shown as means ± SEM. See also Figure S7. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S1 Exploration Behavior in Previously Characterized Roaming/Dwelling Mutants and Serotonin Pathway Mutants, Related to Figure 1 (A) Exploration behavior of animals of the indicated genotypes, shown as means ± SEM and including scores of individual animals (triangles). Asterisk indicates p < 0.01, ANOVA and Bonferroni-Dunn post-hoc test (versus wild-type). (B) Exploration behavior of animals of the indicated genotypes. Assays were performed on larger plates for a shorter duration to distinguish between mutants with high levels of roaming. Asterisks indicate p < 0.01, ANOVA and Bonferroni-Dunn post-hoc test. (C) Exploration behavior of mod-5 mutants, which have increased serotonergic function due to a mutation in the serotonin reuptake transporter. Asterisk indicates p < 0.01, t test. All data are shown as means ± SEM. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S2 Behavioral Parameters in Wild-Type Animals and in mod-1 Mutants, Related to Figure 2 (A) Scatter plot of average speed and angular speed in 10 s intervals for wild-type animals (n = 350 animals, 135,972 bins). Two clusters of data points are observed, (1) high speed/low angular speed roaming intervals and (2) low speed/high angular speed dwelling intervals. (B) Two-state Hidden Markov Model trained on data from 350 adult wild-type animals. Dwell Behavior and Roam Behavior refer to experimentally defined 10 s dwelling intervals and roaming intervals, respectively (see panel A); the Dwell State and Roam State are inferred from the model. (C) Locomotion of one adult wild-type animal on E. coli. 10 s intervals were classified as roaming or dwelling as in (A). Behavioral state refers to the most probable state as determined by the HMM. (D and E) Ccdfs for (D) dwelling state and (E) roaming state durations in mod-1 mutants (dots) fit to a double exponential (pink line); the two exponentials are individually displayed as dashed black lines. w1/w2, weights of each exponential. (F) Histograms of dwelling state durations in wild-type and mod-1 animals. All states are included for both genotypes. (G) Histograms of dwelling state durations in wild-type and mod-1 animals, excluding all dwelling states that are less than 5.5 min long. We chose 5.5 min as a cutoff because this excludes the majority (>97%) of the data points from the faster exponential in mod-1 mutants, allowing separate examination of longer states (confirmed by visual inspection). Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S3 Validation of Cre-Lox Results for Serotonergic Neurons, Related to Figure 3 (A) Cell-specific removal of tph-1 in NSM and HSN increases exploration in the large-plate format used in Figure S1B. Asterisks indicate significant, additive effects of NSM::Cre and HSN::Cre expression (p < 0.001 for NSM and HSN, two-factor ANOVA; no significant interaction between the two factors). (B) Neuronal Cre expression in wild-type animals does not affect exploration behavior. (C) Increased exploration behavior is observed in egl-1 mutants, whose HSN neurons undergo programmed cell death during development. Asterisks indicate p < 0.0001, t test. All data are shown as means ± SEM. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S4 GCaMP5 Fluorescence Changes in Freely Moving Animals Are Not due to Motion Artifacts, Related to Figure 4 (A and B) Locomotion speed plotted as a function of (A) NSM::GCaMP5 fluorescence and (B) NSM::GFP fluorescence. NSM::GCaMP5 fluorescence correlates inversely with locomotion speed across the entire range (r = −0.233, p < 0.0001); NSM::GFP fluorescence does not correlate with locomotion speed. Fluorescence signals were binned (x axis) and corresponding speed values for each bin are shown as a box-plot. Medians are shown as box-plot waist, the top and bottom of the box denote the 25th and 75th centiles, and the bars denote the 10th and 90th deciles. The color of each box indicates the relative density of data points belonging to each bin. Only bins with > 200 data points are shown. (C) Control event-triggered average aligning forward runs to NSM::GFP fluorescence. (D) AIY::GCaMP5 fluorescence is higher during forward run behavior. Data are shown as means of individual animal mean fluorescent signals during forward runs and all other times (control). Asterisk indicates p < 0.001, paired t test. (E) Control event-triggered average aligning forward runs to AIY::GFP fluorescence. (F) An example of AIY calcium imaging in a freely moving animal, monitored using GCaMP5. For (C) and (E), horizontal dashed lines indicate pre-event baseline calcium signals and speed, and data are shown as means ± SEM. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S5 Optogenetic Manipulations of the Serotonergic Circuit, Related to Figure 5 (A) ChR2-mediated activation of all serotonergic neurons decreases speed and increases angular speed in animals that were roaming prior to LED illumination. Controls (gray lines) were identically treated wild-type animals. (B) ChR2-mediated activation of NSM decreases speed, increases angular speed and causes roaming-to-dwelling transitions in animals that were roaming prior to LED illumination. Controls (gray lines) were identically treated wild-type animals. (C) Durations of dwelling states induced by the indicated optogenetic manipulations. Dwelling state durations were measured beginning at the time of LED light termination. Endogenously-generated dwelling states were measured from the same videos. Data are shown as medians ± 95% confidence intervals. Asterisks indicate p < 0.05, Wilcoxon Rank Sum test. (D and E) ChR2-mediated NSM activation does not promote dwelling behavior in (D) mod-1 or (E) tph-1 animals that were roaming prior to LED illumination. Controls (gray lines) were matched mutant animals with no LED illumination. (F) ChR2-mediated activation of mod-1-expressing neurons increases speed and decreases angular speed of animals that were dwelling prior to LED illumination. Controls (gray line) were identically treated wild-type animals. (G) ChR2-mediated activation of AIY and RIF promotes roaming behavior in animals that were dwelling prior to LED illumination. Controls (gray line) were identically treated wild-type animals. (H) Durations of roaming states induced by the indicated optogenetic manipulations. Roaming states durations were measured beginning at the time of LED light termination. Endogenously-generated roaming states were measured from the same videos. Data are shown as medians ± 95% confidence intervals. (I and J) ARCH-mediated silencing of mod-1-expressing neurons promotes dwelling behavior in (K) mod-1 and (L) tph-1 mutants that were roaming prior to LED illumination. Controls (gray lines) were matched mutant animals with no LED illumination. For (A-B), (D-G), and (I-J), data are shown as means ± SEM; asterisks indicate p < 0.01, t test for speed and angular speed; or p < 0.01, chi-square test for fraction of animals roaming. Blue light delivery was followed by 60 s of green light delivery to inactivate ChR2(C128S). Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S6 Behavioral Characteristics of pdf-1 and pdfr-1 Mutants, Related to Figure 6 (A) Touch responses in animals of the indicated genotypes. Animals that had been off food for 30 min were touched 10 times with an eyelash, alternating between anterior and posterior touch. The number of successful responses (i.e., changes in direction upon touch), out of 10 total trials, was recorded for each animal. Means ± SEM are shown for each genotype. (B) Forward speed during roaming and dwelling states for wild-type and pdfr-1 animals. Because state durations differ between genotypes and may influence speed, we only considered speed during states whose durations were present in both genotypes (dwelling states less than 2,000 s; roaming states less than 200 s). Asterisks indicate p < 0.01, Bonferroni-corrected t test. Data are shown as means ± SEM. (C) Complementary cumulative distribution function (ccdf) for roaming state durations in wild-type animals (dots) fit to a double exponential (pink line); the two exponentials are individually displayed as dashed black lines. w1/w2, weights of each exponential (same data as Figure 2B). (D) Ccdf for roaming state durations in pdfr-1 mutants (dots) fit to a single exponential (pink line). (E) Exploration behavior of animals of the indicated genotypes. pdf-1a and pdf-1b are two different splice isoforms of the pdf-1 gene. (F) pdf-1 promoter-driven GFP expression pattern observed in rescued pdf-1; pdf-1::pdf-1b-sl2-GFP animals. (G) Genomic region surrounding pdfr-1 (thick boxes indicate coding exons; thin boxes indicate non-coding exons), region deleted in ok3425, PCR fragments used for transgenic rescue and pdfr-1 distal promoter fragment. (H) Rescue of pdfr-1 mutant phenotype by transgenic expression from PCR fragments depicted in (G). (I) Rescue of pdfr-1 mutant phenotype by transgenic expression of pdfr-1(distal)::pdfr-1-sl2-GFP. For (E) and (H-I), asterisks indicate p < 0.01 by ANOVA and Bonferroni-Dunn post-hoc test. All data are shown as means ± SEM. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S7 Acute and Chronic Manipulations of cAMP Levels in pdfr-1-Expressing Neurons, Related to Figure 7 (A) Exploration behavior of animals of the indicated genotypes, shown as means ± SEM. Asterisks indicate p < 0.01, t test. (B) Durations of roaming states induced by the indicated optogenetic manipulations. Durations of the induced roaming states were measured beginning at the time of LED light termination. Endogenously-generated roaming states were measured from the same videos. Data are shown as medians ± 95% confidence intervals. Asterisk indicates p < 0.01 by Wilcoxon Rank Sum test. (C) Model for relationship between serotonin and PDF neuromodulatory systems. There are two parallel pathways for behavioral state regulation: serotonergic signaling through MOD-1 prolongs dwelling states and PDF signaling through PDFR-1 prolongs roaming states. In addition, serotonin and PDF reciprocally inhibit one another. Cell 2013 154, 1023-1035DOI: (10.1016/j.cell.2013.08.001) Copyright © 2013 Elsevier Inc. Terms and Conditions